WO 94/11734 describes a two-site immunoassay for an antibody using a chemiluminescent label and a biotin bound ligand, said method comprising the steps of (a) mixing the liquid sample with a ligand antigen, antibody or hapten bound to biotin or a functional derivative thereof, an antibody directed against the antibody to be detected bound to paramagnetic particles and a chemiluminescent acridinium compound bound to avidin, streptavidin or a functional derivative thereof to form a solid phase complex, (b) magnetically separating the solid chase from the liquid phase, (c) initiating a chemiluminescent reaction, if any, in the separated solid phase and (d) analysing the separated solid phase for the presence of a chemiluminescent phase, which is indicative of the presence of said antibody in the sample.
The prior art method is particularly suitable for measuring the concentration of specific immunoglobulins in body fluids, such as a specific immunoglobulin selected from the group of IgA, IgD, IgE, IgG, IgM and subclasses thereof.
The prior art method is also suitable for the detection and quantification of the total content of immunoglobulins in a class or subclass, such as IgA, IgD, IgE, IgG, IgM and subclasses thereof.
In a preferred embodiment of the prior art method the formation of the solid phase complex is effected in two steps, viz. a first step wherein the sample is mixed with the biotin bound ligand antigen, antibody or hapten and the antibody bound to paramagnetic particles so as to form a first solid phase complex, and a second step wherein the chemiluminescent acridinium compound is added to the first solid phase complex to form a second solid phase complex.
However, practical use of the prior art method has revealed the fact that in some applications of the method interference from other types of immunoglobulins and/or from other types of immunologically active serum components than the one to be measured occurs leading to errors in the results obtained.
The article xe2x80x9cCapture assay for specific IgExe2x80x9d, V. Olivieri et al, Journal of Immunological Methods, 157 (1993) 65-72 discloses an assay for the measurement of specific IgE in the serum of allergic patients using monoclonal anti-human IgE (coated to the wells of a microtiter plate) and biotinylated allergens in solution. In a single incubation IgE is bound to the solid phase through the Fc fragment and biotinylated allergens react with their specific IgE Fab regions. In a second step, streptavidin-horseradish peroxidase conjugate is added to reveal the amount of biotin fixed on the solid phase. The article studies the interference from high levels of non-specific IgE and from allergen-specific IgG. The assay is found to be unaffected by allergen-specific IgG.
WO 98/16829 discloses an assay, wherein an anti-immnunoglobulin is coupled to a microtiter plate well, which is then washed. The test serum is then added to the well to capture all of total targeted immunoglobulin in the test sample. Then, the biological fluid sample is aspirated, and the microtiter plate is washed. The captured antibody on the microtiter plate is then exposed to biotinylated antigen, the plate is washed, and streptavidin/alkaline phosphatase conjugate is added, and the plate is washed again. The prior art assay may be used to monitor the effect of treatment on H. Pylori infection with standard antibiotic therapy.
A first object of the invention is to provide a method of the type, wherein the targeted antibody is complexed to a Fc directed antibody coupled to a solid particle and to a Fab directed ligand, which does not suffer from the above explained drawback of interference from other components of the test sample.
This first object is achieved with the method of the invention, the essential new feature of the invention being that an additional sequence of separation and washing of the intermediate solid phase complex consisting of particle with reactant antibody and sample antibody is carried out prior to addition of ligand.
The method of the invention is based on the recognition that by introducing such an additional sequence of separation and washing, potentially interfering excess material from the liquid sample as well as potentially interfering excess component (ii) may be removed from the method thus eliminating the risk of interference of the said factors in subsequent steps. It has surprisingly been found that the additional sequence of separation and washing has reduced substantially and in some circumstances eliminated the technical problem of interference between different types of immunologically active serum components.
The reduction of the interference obtained involves a number of technical advantages. In particular, it allows a more precise measurement of problematic sera having a difficult and unpredictable ratio of mixture of antibodies. Furthermore, falsely positive identifications of an antibody may be avoided. Also, the reduction of interference allows precise measurements to be made in a wider antibody concentration range than with prior art methods.
A second object of the invention is to provide a method, which is capable of evaluating and/or predicting the effect of a Specific Allergy Vaccination (SAV).
This second object is obtained by the nature and the temporal development of the interference between different types of immunologically active serum components, e.g. antibodies, are used as parameters for evaluating/predicting the effect of a Specific Allergy Vaccination treatment. Thus, it has surprisingly been found that the said parameters hold valuable information about the immunological status of a person as well as the response of a person to a selected treatment scheme.
A third object of the invention is to provide a method of evaluating the immunological status of a subject.
The third object of the invention is obtained by the nature and the temporal development of the interference between different types of immunologically active serum components, e.g. antibodies, are used as a parameter for evaluating the immunological status of a subject, in particular evaluating/predicting the effect of allergy treatment, allergy vaccination treatment or Specific Allergy Vaccination treatment. Thus, it has surprisingly been found that the said parameter hold valuable information about the immunological status of a person as well as the response of a person to a selected treatment scheme.
A fourth object or the invention is to provide a method of evaluating the effect of allergy treatment of a subject.
The fourth object of the invention is obtained based on the recognition that the measurement obtained with the subassays 1, A and C, i.e. a measurement, which is carried out in the presence of interfering factors in the sample, is particular useful for evaluating the effect of allergy treatment, allergy vaccination treatment and Specific Allergy Treatment (SAV). Thus, the measurement obtained with this method is obtained under conditions, which correspond to in vivo conditions, and hence is a more physiological and clinical relevant measurement for evaluating treatment effects.
A preferred embodiment of the first aspect of the invention is characterized in that component (iii) of step (rxe2x80x2), (r) or (rxe2x80x3) and component (iv) of step (sxe2x80x2), (s) or (sxe2x80x3), respectively, are added in one operation.
A first alternative embodiment of the first aspect of the invention is characterized in that the three-component solid phase complex formed in step (rxe2x80x2), (r) or (rxe2x80x3) prior to subjecting it to step (sxe2x80x2), (s) or (sxe2x80x3), respectively, is washed to remove non-complex bound compounds.
Specific allergy vaccination (SAV), formerly known as specific Immunotheraphy or Hyposensitization, has been used for the treatment of Type 1 IgE mediated allergic disease since the beginning of this century.
The general benefits obtained through SAV are: a) reduction of allergic symptoms and medicine consumption, b) improved tolerance towards the allergens in the eyes, nose and lungs and c) reduced skin reactivity (early and late phase reactions).
The basic mechanism behind the improvement obtained by SAV is unknown, but a number of common features can be extracted from the numerous SAV studies performed in the last decades: 1) the amount of total IgE is unchanged during the treatment period, 2) the amount of allergen specific IgE increases transiently during updosing, then it falls back to the initial (pretreatment) level, 3) the epitope specificity and affinity of IgE remains unchanged, 4) allergen specific IgG, in particularly IgG4, raises sharply during SAV, 5) a new Th0/1 response is apparently initiated and 6) the Th2 response seem unchanged. There is no correlation between the effect induced by SAV and the onset of specific IgG.
SAV induces a new immune response which matures during the treatment period (Th0/1 T-cells are recruited, an allergen specific IgX (X may be A1, A2, G1, G2, G3, G4, M or D) is initiated). As the affinity (or amount/affinity) of the new antibody response, IgX, has matured, IgX may compete efficiently with IgE for the allergen(s), inhibiting the xe2x80x9cnormalxe2x80x9d Th2 based allergic response characterised by the cross-linking of receptor bound IgE on the surface of mast-cells and basophils. Hence, clinical symptoms will gradually be reduced.
The present invention is based on the hypothesis that if one measures the amount or specific IgE in the presence and absence of competing compounds (IgX and/or any other interfering substance), a measure for the competitive capability of immune responses in the individual patient may be calculated and this measure would correlate with an appropriate effect parameter.
Most prior art xe2x80x9cquantitativexe2x80x9d IgE assays measure IgE in the absence of competing substances. The present invention describes the measurement of IgE in the presence and absence of any (serum originating) competing substance. Thus, the methods referred to in FIGS. 2a-c measure IgE in the absence of competing agents, whereas the method defined in step (ixe2x80x2), (i), (ixe2x80x3), (yxe2x80x2), (y) and (yxe2x80x3), respectively, measure IgE in the presence of competing agents.
It is believed that the mode of action of the method of the invention may be explained as follows: If SAV induces a response which competes with IgE for the binding of the allergen it should be possible to measure the effect of the treatment by comparing IgE determined by the above stated two methods, if the two methods produce the same result no interfering substance has been induced and there is no effect. Furthermore, if the measurement of the latter method (in the presence of competing substances) is lower than the measurement of the former method (in the absence of competing substances) a competing response has been mounted and there is an effect of the treatment.
In the second aspect of the invention, two subassays are used in the method, viz. subassays, wherein the reaction between sample antibody and allergen is effected in the absence (subassay (hxe2x80x2), (h), (hxe2x80x3), (xxe2x80x2), (x) and (xxe2x80x3)) and presence (subassay (ixe2x80x2), (i), (ixe2x80x3), (yxe2x80x2), (y) and (yxe2x80x3) of the other sample constituents, respectively.
In subassays (h) and (hxe2x80x3), the chemiluminescent label may be an acridinium compound.
A preferred embodiment of the subassays (hxe2x80x2), (h) and (hxe2x80x3) is characterized in that in step (axe2x80x2), (a) or (axe2x80x3) components (i), (ii) and (iii) are mixed in one operation (FIG. 3b).
A first alternative embodiment of the subassays (hxe2x80x2), (h) and (hxe2x80x3) is characterized in that in step (axe2x80x2), (a) or (axe2x80x3) components (i) and (ii) are mixed in a first operation and that component (iii) is added in a second operation (FIG. 3c).
A second alternative subassays (hxe2x80x2), (h) and (hxe2x80x3) is characterized in that in step (axe2x80x2), (a) or (axe2x80x3) components (i) and (iii) are mixed in a first operation and that component (ii) is added in a second operation (FIG. 3a).
A preferred embodiment of the second aspect of the invention is characterized in that step (iaxe2x80x2), (ia), (iaxe2x80x3), (yaxe2x80x2), (ya) or (yaxe2x80x3) is carried out by mixing components (i) and (ii), then adding component (iii), and finally adding component (iv), if added.
Another preferred embodiment of the second aspect of the invention is characterized in that step (iaxe2x80x2), (ia), (iaxe2x80x3), (yaxe2x80x2), (ya) or (yaxe2x80x3) is carried out by mixing components (i), (ii) and (iii), and then adding component (iv), if added.
A further preferred embodiment is characterized in that the comparison of step (jxe2x80x2), (j), (jxe2x80x3), (zxe2x80x2), (z) or (Zxe2x80x3) is carried out by calculating the ratio of the measurements of the said two steps. Alternatively, the comparison is carried out by calculating the difference between the two measurements.
Still a further embodiment of the invention is characterized in that the comparison of step (jxe2x80x2), (j), (jxe2x80x3), (zxe2x80x2), (z) or (zxe2x80x3) is carried out at a number of points in time at the start of and during the treatment period, and that any temporal change, which may be observed, is used as a basis for evaluating and/or predicting the effect of the treatment.
The third aspect of the invention is based on the same recognitions and hypothesis as the second aspect, the difference being that the third aspect is not limited to any specific immunoassay procedure. Also, according to the third aspect the assay may be used to predict the effect of all types of allergy treatment.
The interrelating of the two measurements obtained may be carried out by calculating the ratio of or the difference between the measurements.
The term xe2x80x9callergy treatmentxe2x80x9d means any treatment of allergy. The term xe2x80x9callergy vaccination treatmentxe2x80x9d means any vaccination treatment of allergy. The term xe2x80x9cSpecific Vaccination Treatment (SAV)xe2x80x9d is described above.
In the present invention the expressions xe2x80x9cthe antibody of the samplexe2x80x9d and xe2x80x9csample antibodyxe2x80x9d may mean a specific immunoglobulin, preferably a specific immunoglobulin from the classes IgA, IgD, IgE, IgG, IgM and subclasses thereof. In general, the said antibody may be any blood serum or plasma component, which is capable of interfering specifically with the interaction between immunoglobulines and a ligand in the form of an antigen, an antibody or hapten, e.g. enzyme inhibitors and receptors.
The term xe2x80x9cliquid samplexe2x80x9d means any liquid or liquefied sample, including solutions, emulsions, dispersions and suspensions. The sample may be a biological fluid, such as blood, plasma, serum, urine, saliva and any other fluid, which is excreted, secreted or transported within a biological organism.
The expression xe2x80x9cligand in the form of an antigen, an antibody or a haptenxe2x80x9d may be any immunologically active substance. xe2x80x9cAntigenxe2x80x9d may be an allergen, e.g. pollen from trees, grass, weeds etc., mould allergens, allergens from acarids (mites) and animals, such as cat, dog, horse, cattle and bird, allergens of stinging insects and inhaled allergens originating from insects, and food allergens; xe2x80x9cantibodyxe2x80x9d may be a monoclonal or polyclonal antibody, including recombinant and fragmented antibodies; and xe2x80x9chaptenxe2x80x9d may be carbohydrate moieties or fragments thereof, enzyme inhibitors or drugs, e.g. penicillin or a derivative thereof.
The expression xe2x80x9clabelled ligandxe2x80x9d means any ligand comprising a labelled atom or part, e.g. a radioactive atom label.
The expressions xe2x80x9clabel compoundxe2x80x9d means any suitable label system conventionally used in immunoassays comprising luminescent labels, chemiluminescent labels, enzyme labels, radioactivity labels, fluorescent labels, and absorbance labels.
The term xe2x80x9ccarrierxe2x80x9d means any solid support, which may be used in an immunoassay, e.g. a microtiter plate, a particle, a tube, a sponge of a polymer material (matrix), etc.
The term xe2x80x9csolid particlexe2x80x9d means any particulate matter, which can be suspended in a liquid, e.g. glass beads, metal, e.g. iron particles, particles of polymer material, etc.
The separation of the solid phase complex from the liquid phase may, depending on the type of solid particle used, be carried out by i.a. magnetic separation, filtration, sedimentation, centrifugation, chromatography, column chromatography.
The term xe2x80x9csolid paramagnetic particlexe2x80x9d means any paramagnetic particle, which may be dispersed or suspended in a liquid medium, e.g. xe2x80x9cBiomagxe2x80x9d particles (iron oxide particles coated with amine terminated groups) sold by Advanced Magnetics Inc., U.S.A., and xe2x80x9cDynabeadsxe2x80x9d (iron oxide covered with a polymer) sold by Dynal A. S., Norway.
The term xe2x80x9creactant antibodyxe2x80x9d means any antibody or other biospecific reagent capable of reacting with the sample antibody comprising monoclonal and polyclonal antibodies, including recombinant and fragmented antibodies, e.g. a monoclonal antibody, xe2x80x9cMAb A 5697-1A3(920325) supplied by BioInvent International AB, Sweden, xe2x80x9cProtein Axe2x80x9d or xe2x80x9cProtein Gxe2x80x9d supplied by Sigma Chemical Company, Saint Louis, USA.
The chemiluminescent compound is preferably an acridinium compound, such as N-hydroxy-succinimide dimethylacridiniumester (NHS-DMAE). Avidin/streptavidin and DMAE may be coupled according to the methods of Weeks et al., Clinical Chem., 29, 1474-1479 (1983). Other examples of chemiluminescent compounds suitable for use in the present invention are luminol, lucigenin and lophine.